Dietary Supplements - American Chemical Society

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on November 30, 2015 | http://pubs.acs.org Publication Date: September 1, 2008 | doi: 10.1021/bk-2008-0987.ch018

Chapter 18

PurePowder®: A New Process for Sterilization and Disinfecting of Agricultural and Botanical Herbal Products 1

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Kan He , Zhongguang Shao , Naisheng Bai , Tangsheng Peng , Marc Roller , and Qunyi Zheng 3

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Department of Research and Development, Naturex, Inc., South Hackensack, NJ 07606 SGS, 291 Fairfield Avenue, Fairfield, NJ 07004 Naturex, Inc., 84911 Avignon Cedex 9, France Kent Financial Service, Inc., 211 Pennbrook Road, Far Hills, NJ 07931 2

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Herbal powders comprise a large portion of the botanical products sold in the dietary supplement market. These powders are generally contaminated with microbial organisms found in botanical raw materials either harvested from farms or collected from the wild. Currently, ethylene oxide and gamma irradiation are the dominant modes used to sterilize botanical powders. However, these technologies have recently been subject to safety concerns due to their potentially harmful effects on human health. We report here a new process as an alternative to sterilizing and disinfecting botanical powders using hydrogen peroxide. The mechanism of the sterilization and disinfection is the use of hydrogen peroxide whereby contaminated products are brought into contact with nascent oxygen, resulting in the oxidization and destruction of the contaminating microorganisms.

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© 2008 American Chemical Society

In Dietary Supplements; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


265 Botanical products, including potentially contaminated herbs, received a substantial boost when the U.S. Congress passed the Dietary Supplement and Health Education Act in 1994 (1-3). Herbal products that are sold in the marketplace consist of either a powder form, derived directly from the ground plant, or an extract. There is less of a concern for microbial contamination on the extract because sterilization has taken place during extraction. Plant powders normally carry a great number of bacteria and molds that often originate in soil. Practices of harvesting, handling, and production may cause additional contamination and microbial growth. Government regulations require low microbial limits for final herbal products that are usually subjected to disinfectant treatments by means of fumigation with ethylene oxide, thermal treatment with steam, and irradiation withγ-raysor high-energy electrons. Both gamma and electron-beam radiation processes are used commercially for sterilization (4). The process is generally considered safe when carried out under controlled conditions and in suitable facilities. However, today's consumers are becoming increasingly reluctant to accept a product that has been irradiated. For safety concerns, herbal supplements are not allowed to be irradiated in the European Union. However, certain constituent of herbal supplements may legally be irradiated provided that the final products indicate their presence on labeling. Ethylene oxide (ETO) is also used by the herbal industry as a sterilant because of its potency in destroying microorganism. Safety concerns are due to the flammable and explosive properties of ETO, as well as it's being a probable human carcinogen. Furthermore, toxic emission, toxicity of by-product ethylene chlorohydrin, and residues of ETO may present health hazards to people. The use of ETO has been forbidden within countries of the European Union and in Japan. Super-heated steam sterilization has the advantage of being an established, effective and well-understood technology. Steam is incompatible with thermo-labile components that comprise a large portion of the materials used in herbal products. To heat-resistant (thermophyllic) bacterial strains, steam is less effective. Hydrogen peroxide (H 0 ) has been used in many industries as a disinfectant. The hydrogen peroxide solution is a germicide that is active by virtue of the fact that it releases nascent oxygen. It is a very short acting element since this release occurs rapidly. Nascent oxygen is oxygen in atomic form characterized by excessive chemical activity. This form is known to be one of the most reactive chemical species, weaker than the element fluorine and hydroxyl radical, but stronger than ozone, perhydroxyl radical, permangnate, hypobromous acid, chlorine dioxide, chlorine, etc. Hydrogen peroxide is very unstable and is easily decomposed to form oxygen and water by increasing the temperature or by reducing agents. Thus, the use of hydrogen peroxide is predicted as safe, effective, economical and environmentallyfriendly.By using hydrogen peroxide as an oxidizer, we have developed a new technique, the PurePowder® process, which is based on an oxidation process to achieve 2

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266 complete elimination of microbial contamination (5). The process reported herein can be an alternative method to the abovementioned methods.


Materials and Procedures

Test for Residual Hydrogen Peroxide Hydrogen peroxide, at a concentration of 35%, was purchased from Arkema, Inc., (Philadelphia, PA) and was used in the entire experiment. Residual hydrogen peroxide was tested using EM Quant Peroxide Test kits (EM Science, Gibbstown, NJ). 0.5-1 g of powder was added into 2-4 mL of distilled water (or ethanol or acetone, in the case of organic solvent). The solution was then mixed and sonicated for 15 minutes at room temperature. After sonication, the solution was filtered through a 0.45 jum filter for further use. For this test the strip was dipped into the solution for 1 second. The test strip was removed, the excess liquid was shaken off and the reaction zone was compared with the color scale after 15 seconds. In the case of organic solvents, the test strip was dipped into the solution for 1 second. The test strip is moved slightly to andfrofor 3-30 seconds until the solvent has evaporatedfromthe reaction zone. The rest of the procedure is the same as aqueous solutions.

Test for Active Components and Chemical Profiles The active marker components in herbal products were tested using high performance liquid chromatography (HPLC) technique and run on a Hewlett Packard model 1100 equipped with an autosampler, UV/VIS detector, and Hewlett Packard ChemStation software. HPLC conditions included the use of a Phenomenex, Prodigy ODS (5 µm, 4 ID x 125 mm) column or equivalent C-18 column. Gel permeation chromatography (GPC) was performed on PL aquagelOH 30, (8. µm, 7.8 ID x 30). The proton nuclear magnetic resonance (NMR) spectra were acquired on a Varian Unity Inova 400 system.

Test for Microorganisms Total aerobic count (TAC) and yeast & mold (Y&M) count were tested by using the BioMérieux Bactometer method. One gram of sample was added to 99 mL of phosphate dilution buffer (pH 7.2 ±0.2) (for 1000 cfu/g cutoff), or 10 gram of sample was added to 90 mL of phosphate dilution buffer (for 100 cfiu/g



267 cutoff). The mixture was shaken to mix well and a pipette was used to transfer 0.1 mL of this dilution into duplicated wells of a prepared General Purpose Media Bactometer module (for TAC) and a prepared Yeast and Mold Media Bactometer module (for Y&M). The TAC module was incubated in a 35 °C bactometer chamber for 24 hours and the Y & M module was incubated in a 25 °C bactometer chamber for 48 hours. The growth of the microorganism was monitored by the Bactometer Processing System. The presence or absence of Salmonella and Escherichia coli was tested following the procedures published in USP 24. The Enterobacerial Count was tested following the procedures published by Pharmacopeial Forum (Vol. 25 (2), page 7761).

General Procedure for PurePowder Process To the herbal crude powder, about 2-15% of the weight of herbal powder of hydrogen peroxide (H 0 , 35% concentration) was applied. After the dispersion of the H 0 solution, the mixing powder was heated for about 10-30 minute at 60-90°C. Samples were taken for microbial and hydrogen peroxide residue testing. 2

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Anti-DPPH Test The solution of a,a-diphenyl-p-picrylhydrazyl (DPPH) was purchased from Sigma Chemical Co. (St. Louis, MO). A 12.2 mg of DPPH solution was mixed into 100 mL of methanol as stock solution and was stored in a refrigerator and kept from light before using. About 1-2 g of herbal powder was weighed and sonicated in 25 mL of methanol for 1 hour. DPPH stock solution was added to the methanol extract to make the final DPPH concentration of 0.5 mM. The mixture was shaken vigorously and left to stand in the dark at room temperature for 30 minutes. The absorbance of the resulting solution was measured using a spectrophotometer read at 517 nm (Cary 300 Bio UV-visible spectro photometer). The percentage of anti-DPPH was calculated as the difference of the absorbances at 517 nm of DPPH and sample and divided by absorbance of DPPH.

Results and Discussion This sterilization process has proven to be effective in the reduction of microbial contamination from a total aerobic count levels from as high as 900,000 to as low as less than 10 cfii/g. Similarly, the yeast and mold count is reduced from as high as 400,000 to less than 10 cfb/g. The process also



268 completely eliminates Escherichia coli and Salmonella contamination (Table I). The hydrogen peroxide residue was below the testing limit, which was less than 0.5 ppm after the process was completed. It is generally recognized that, since most of the microorganisms contaminating herbal powders reside on their surfaces, the inner parts do not have to be exposed to heat, gas or radiation for decontamination. In the current method, hydrogen peroxide is applied onto the surface of plant material and the reaction conditions are adjusted to decompose the hydrogen peroxide to release the nascent oxygen or hydroxyl radical, which chemically react with the surrounding molecules causing damage to biological cells, including those of the contaminating microorganisms or insects. Microorganisms are more sensitive to be attacked by hydrogen peroxide while the chemical components inside the plant cell are relatively stable from being oxidized. For example, Eleutherococcus senticosus was used as "adaptogen" in the US dietary supplemental market. Phenylpropane derivatives, eleutherosides B and E serve as markers in the standardized Eleuthero product. It was observed that the contents of eleutherosides B and E before and after sterilization with H 0 in Eleuthero powder were similar and the HPLC profiles were identical. This indicates that the chemical compositions have not been significantly changed through this sterilization process (Figure 1). 2

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The US native plant, Echinacea purpurea, gained worldwide popularity as an immune stimulant. Phenolic compounds, chicoric acid and caffeoyal tartaric acid are two major components used as markers in the standardized Echinacea purpurea product. Similar to Eleuthero powder, no significant changes were found in the contents of chicoric acid and caffeoyal tartaric acid in Echinacea powder before and after H 0 processing (Figure 2). Valeriana officinalis has a long history of being prescribed for sleep disorder. Sesquiterponoids, valerenic acids are the markers of quality control of Valerian products. No significant degradation was detected in the contents of valerenic acids through H 0 treatment (Figure 3). The above examples demonstrate that hydrogen peroxide attacks the microorganism on the surface of the plant more than the chemical components inside the plant. Similar examples containing varieties of natural products can also be found without significant degradation. These included triterpene glycosides in ginsneg, black cohosh, astragalus, tetraterpenoid (3-carotene in carrot, sequiterpene parthenolide in feverfew, iridoids harpagoside in devil's claw, flavonoids in ginkgo biloba, milk thistle, passion flower, anthraquinones pseudohypericin, hypericin in St. John's wort, fatty acids in saw palmetto, maca, alkaloids in goldenseal and yohimbe, etc. In some cases there were some color changes, which were noticed in the current sterilization process if the condition was not well controlled, i.e. over using hydrogen peroxide and overheating. In the case of powders milled to a very fine mesh, some degradation of chemical components was observed with the probable explanation that due to a fine 2

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Table 1. Results of Microbial Analysis on Some Examples of Herbal Powder before and after Hydrogen Peroxide Processing


Herbal Name

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Y&M

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TAC

Black cohosh root & rhizome powder (Cimicifuga racemosa)

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>10,000

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100,000

P

>20,000

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20,000

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220,000

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10,000

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25,000

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116,000

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2,000

After

32,000

After

400,000

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4,000

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Dietary Supplements - American Chemical Society

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